Lab 8. Lymphatic System

Objective

The student should become familiar with the widespread distribution of the
lymphatic system throughout the body, recognize and distinguish the different
lymphatic organs and tissues and understand the arrangement of cells within
them in relation to their functions.

The lymphatic system consists of lymphatic vessels and lymphatic tissue. Lymphatic
capillaries begin as blind pouches in the connective tissue of nearly all organs.
Lymphatic vessels have not been found in brain, spinal cord, bone marrow, cartilage,
eyeball, or inner ear, even though lymphocytes may be found in some of these
tissues. The walls of lymphatic vessels are composed of endothelial cells and
a few collagenous fibers. When dilated, lymphatic capillaries have a greater
and more variable diameter than blood capillaries. Lymphatic capillaries drain
into larger collecting vessels which contain paired valves. These vessels have
thin irregular walls and pursue an erratic course toward the nearest regional
lymph nodes, where some pierce the convex surface of a node as afferent lymphatics.
Their contents then enter lymphatic sinuses within the node. Efferent vessels
emerge at the concave or hilar surface of the lymph nodes. When lymphatic vessels
reach about 200 µm in diameter, the wall may be divided into an intima,
media and adventitia. The large vessels eventually drain into the thoracic duct
or the right lymphatic duct.

Lymphatic tissue is made up of a framework of reticular fibers which are produced
by reticular cells and which support lymphocytes, macrophages, and related cells.
Lymphatic tissue may be found either as scattered foci of cells, as dense nodules
within connective tissue (especially in the gut as tonsils or Peyer's Patches),
or as aggregations of lymphoid cells enclosed within a capsule such as the lymph
nodes, spleen and thymus. Lymphoid tissue can be described as:

A. Non-Encapsulated Lymphatic Tissue

Examine slide #58, a section of human appendix, by reversed
ocular. A central lumen bordered by pale-staining epithelial cells should
be visible. Surrounding the lumen is a dense blue mass of lymphocytes.
Within this mass lighter regions may seen. These are the germinal centers
of lymph nodules. On some slides nodules are hard to identify. On slide
#58 even, which is stained with MEA, segments of venules with high
endothelium may be seen at the periphery where there are fewer lymphocytes.
Note that the endothelium does not have the typical squamous appearance
but is closer to cuboidal. Lymphocytes may be seen crossing this endothelium.
Examine the diffuse lymphatic tissue and look for eosinophils, plasma
cells (large cells with eccentric nuclei and blue cytoplasm) and lymphocytes.

Peyer's patches are examples of non-encapsulated lymphoid tissue found
along the ileum opposite the attachment of the mesentery. The nodules are usually
pear-shaped structures with their apices just below the intestinal epithelium
and 9 or more nodules aggregate. They will be seen in a later laboratory (G.I.
Tract 2).

B. Encapsulated Lymphatic Tissue: Tonsils

The tonsils are accumulations of diffuse, nodular lymphatic tissue found in
the wall of the naso and oropharynx but are partially encapsulated. On their
outer surface, the tonsils are covered by epithelium which forms infoldings
or crypts. The internal surface is partially covered by a poorly organized capsule
which sends connective tissue septa into the parenchyma. The three tonsils may
be identified by the nature of their epithelium and the arrangement of the crypts.
The pharyngeal tonsil is usually covered by pseudostratified epithelium
which is invaginated in a series of folds often associated with mucous or seromucous
glands. The palatine tonsils are covered by a stratified squamous epithelium
and contain relatively long branched crypts. The lingual tonsil located
at the base of the tongue is covered by stratified squamous epithelium and usually
has a long unbranched crypt. Skeletal muscle and mucous glands of the tongue
may be found closely apposed to this tonsil.

Examine the palatine tonsil (slide #31). Locate the epithelium
(Stratifiedsquamous) and identify crypts. Next, identify
nodules. A primary nodule is a uniform mass of small and medium size lymphocytes
in a delicate framework of reticular fibers. They are characteristically
found in lymph nodes of fetuses and neonates. In adults, most nodules
are secondary nodules and contain a pale staining central portion called
a germinal center composed of large differentiating lymphocytes
(transitional cells, plasmablasts and plasma cells, reticular cells
and macrophages. Some macrophages may be recognized by the
presence of ingested cellular debris (e.g., nuclei). Students need not
distinguish the various cell types present. A corona of small lymphocytes
(mainly B-cells) makes up the outer portion of each nodule. Macrophages
may be recognized as large cells containing fragments of dead lymphocytes.

Lymph Nodes

These are encapsulated bean-shaped structures of lymphatic tissue located
in many regions of the body. They lie along the course of lymphatic vessels
and vary in size from a few millimeters to more than a centimeter in length.
Important nodes are present in the axillae, groin, and neck which are
frequently swollen in patients with ongoing infections. The lymph node
is embedded in fatty connective tissue and is covered by a fibrous
capsule. Examine slide #33 (lymph node) from both odd
and even boxes. Find the c.t. capsule. Although they are unlikely
to be seen on your slides, afferent lymphatics perforate
the capsule and deposit lymph and cells into the subcapsular sinus,
a clear space visible just beneath the capsule and separating it from
the densely basophilic cortical region. Branching inward from the subcapsular
sinus are the cortical sinuses which lead the lymph through the
cortex to the medulla, an irregular and more eosinophilic
region. Identify cortical sinuses (e.g., branches leaving the subcapsular
sinus). Try to identify endothelial cells and reticular fibers spanning
the lumen. Beneath the subcapsular sinus are lymph nodules located
in the outer part of the cortex. Their structure and composition is the
same as that described above for the tonsils.

Somewhat deeper to the area containing nodules and not physically demarcated
from them is the paracortical area which is composed primarily of sheets
of lymphocytes. Most small lymphocytes found in the paracortical areas are thymus-dependent
(T-cells). These cells arrive either via afferent lymphatic vessels or
high endothelial venules which can be seen in this region. B-lymphocytes (B-cells)
predominate in the outer cortex especially in the corona of nodules. Lymphocytes
may leave the lymph node by passing into the sinuses (subcapsular, cortical
and medullary) and then into efferent lymphatic vessels in the hilus. Lymphocytes
pass through several nodes before entering the thoracic duct and eventually
the large veins (subclavian) of the neck.

Locate the medullary region of the lymph node consisting of irregular
anastomosing cords of cells (medullary cords) consisting of reticular
cells,macrophages, plasma cells, and lymphocytes. Between cords
are the medullary sinuses. Due to the shape of the lymph nodes,
some sections contain the central region and may lack the hilar
region, and have varying amounts of medullary tissue. Examine the reticular
fiber framework of a lymph node on slide #34 to understand
the supporting architecture. Note sinuses and their structure.

Spleen

The spleen is the largest mass of lymphatic tissue in the body. It consists
of cells and vessels contained within a capsule lined by mesothelium, from which
trabecula enter the splenic parenchyma. The parenchyma is supported by a framework
of reticular fibers. The spleen consists of two types of parenchymal tissue,
the white and red pulp. The white pulp is composed of elongated cords of compact
lymphatic tissue containing nodules. The red pulp is composed of pulp cords
and splenic sinusoids (sinusoid is another term for blood sinus).

Examine the spleen slide #35, odd and even and locate
the capsule covered by a mesothelium (may have been damaged). Collagen,
elastic and a few reticular fibers as well as some smooth muscle cells
make up the capsule and the trabeculae (projections from the capsule
into the organ). The arrangement of the fibers permits the spleen to expand
passively and to serve as a small temporary reservoir for blood. Note
that the trabeculae which originate at the hilus contain and support trabecular
arteries and veins which enter and leave the organ at the hilus. Trabeculae
from other areas of the capsule do not contain vessels. Arteries branching
out of the trabeculae are immediately surrounded by lymphocytes forming
the white pulp.

Examine the white pulp (blue stained regions) and note that
the lymphocytes make up the majority of the cells. A dense lymphatic
collar of mainly T-cells surrounds the central (or follicular)
arteriole. This collar is called the periarterial lymphatic sheath
(PALS). Despite its name the central arteriole is usually on one side
of the white pulp. Although not often visible, nodules are scattered along
the length of the PALS. At 10x, the dense blue collar is seen to have
a diffuse edge, the marginal zone, which is highly vascular (best
seen on the injected specimen) and is a zone of B and T cell interaction
containing many antigen presenting cells. Many branches of the central
arteriole ramify and are believed to end blindly at the marginal zone.

Branches of the central arteriole pass from the white pulp into the
red pulp and branch to form penicillar arteries or penicilli.
A penicillus has three sections; the first is a straight vessel
called the artery of the pulp or pulp arteriole. The pulp arteriole
branches to form many smaller arterioles some of which are sheathed
arterioles. These have a slit-like lumen surrounded by macrophages
which make up the peri-arteriolar macrophage sheath (PAMS). The
sheathed arteriole branches to form the terminal arterial capillary,
which empties into either a pulp cord (open circulation) or a sinusoid
(closed circulation). The smaller branches of the penicillus
are difficult to identify, but students should be able to identify pulp
arterioles. Blood vessels may be easier to find on slide #35, even.

Most of the terminal vessels end openly (open circulation) delivering
blood into the pulp cords which consist of reticular c.t. containing
reticular cells, macrophages, plasma cells, lymphocytes and elements of
the blood. Some terminal arterioles may connect directly with venous sinusoids
(closed circulation) as shown in the diagram).

Blood delivered to the pulp cords eventually enters venous sinusoids
which are lined by elongated endothelial cells separated by slits. Their basement
membrane is discontinuous and they are lined on their outer surface by reticular
cells and supported by elongated reticular fibers. Distinguish between sinusoids
and pulp cords. The sinuses may be easier to distinguish on slide #35, odd,
(spleen). The venous sinusoids empty into pulp veins which then form trabecular
veins and exit from the spleen at the hilus.

Thymus

The thymus is bilobed and each lobe is segmented into lobules. The parenchyma
is arranged in cortical and medullary components. There are no lymph nodules,
sinuses or afferent lymphatics although efferent lymphatics are present. The
major cellular components of the thymus, thymocytes, are cells which are morphologically
indistinguishable from circulating small lymphocytes. Epithelial reticular cells
are pleomorphic, stellate, non-phagocytic cells which seem to be supportive
in function and are held together by desmosomes. They replace the fibroblastoid
reticular cells found in other lymphoid organs. Other epithelial cells in the
medulla have the ultrastructure of secretory cells. Although different epithelial
cells throughout the thymus appear alike by light microscopy their ultrastructure
and function varies.

Examine slide #37, (even boxes) (thymus of a young person) by
reverse ocular and note its lobular nature. The thymus consists of a central
core of lightly-stained medullary tissue with numerous lateral
extensions, each of which is capped by a layer of dark blue staining cortical
tissue. Each medullary extension plus its cortical tissue makes up
a lobule. Cortical tissue may appear to completely surround medullary
areas, but this appearance is due to the angle of section. Between lobules
are connective tissue septa, extensions of the capsule containing
arterioles and veins. Macrophages, granulocytes and plasma cells are occasionally
seen in the septa.

Scattered among the densely packed lymphocytes of the cortex
are clear patches which, under high power, can be identified as large
macrophages containing fragments of dead cells. The supporting epithelial
reticular cell framework of the cortex is obscured by the densely packed
lymphocytes. However, at 40x the large oval or elongated, pale blue nuclei
of epithelial reticular cells can be seen underlying the field
of small lymphocytes, especially at the less dense border of the cortex.
Macrophages, pericytes or epithelialcells
are found surrounding the vessels of the cortex, which are generally smaller
than those of the medulla. The sheath of reticular-epithelial cells surrounding
blood vessels is part of the thymus-blood barrier.

Examine the medulla on slide #37 (even) and note the thymiccorpuscles (also called Hassall's corpuscles). Although
these structures are of value in identifying the thymus, their physiological
significance is not known. They are composed of degenerating epithelial
cells arranged in layers around a center of necrotic and hyalinized material.
When stained with H & E, thymic corpuscles look eosinophilic, but
with the MEA stain, (#37 even) they appear pale grey. Examine some
of the smaller blood vessels and note the presence of a sheath of epithelial
cells surrounding them. Blood vessels in the cortex have the same arrangement
but are harder to see. Numerous blood vessels are present at the cortico-medullary
junction.

Examine the thymus of an adult (slide #37, odd boxes) which
is stained with H & E. With age, there is a heavy deposition of fat
associated with the connective tissue and a marked involution (diminution)
of the cortex. Note the abundant fat cells beneath the capsule
and in associated septa. The distinction between cortex and medulla
may be difficult to recognize in sections stained with H & E. However,
attempt to distinguish medulla by: (1) the presence of the markedly eosinophilic
Hassall's corpuscles; (2) the lighter stain of the medulla due to the
less densely packed lymphocytes; (3) the blood vessels rimming the medulla
at the cortico-medullary junction.